Ubiquitin is a 76-residue protein that exists in cells either free or conjugated to many other proteins. Selective degradation of proteins by the ubiquitin/proteasome-dependent pathways plays a role in a multitude of biological processes, including cell growth and differentiation, signal transduction and responses to stress. One ubiquitin/proteasome-dependent proteolytic system is the N-end rule pathway, identified by the laboratory in 1986. The N-end rule relates the in vivo half-life of a protein to the identity of its N-terminal residue. The N-end rule pathway is present in all organisms examined, from mammals to bacteria. Studies supported by the present grant ("Mechanics and Functions of the N-end Rule Pathway", DK39520), currently in its 11th year, have yielded significant insights into the N-end rule's functions and mechanisms, as described in the Progress Report. The objective of the research described in this renewal application is to advance the understanding of the N-end rule pathway and related aspects of the ubiquitin system. Specific aims are: 1) Regulation of peptide import by the N-end rule pathway: biochemical and genetic studies; 2) A fusion-based screen for physiological substrates of the N-end rule pathway in S. cerevisiae; 3) Isolation and analysis of regulators of the N-end rule pathway; 4) Screens for mutants whose viability requires the presence of the N-end r; 5) The use of suppression subtractive hybridization to identify new functions of the N-end rule pathway; 6) Analysis of a mitochondrial function of the Ntalp N-terminal amidase in S. cerevisiae; 7) A biochemico-genetic approach to identification of physiological N-end rule substrates; 8) A "double-headed" inhibitor of the N-end rule pathway; 9) The "two-ubiquitin" technique and its application to the problem of cotranslational proteolysis; and 10) Biochemical and genetic dissection of the UBRI-encoded Nrecognin and Ubr2p, a recently identified homolog of Ubrlp in S. cerevisiae.